IL-1 receptor antagonists medicaments

ABSTRACT

Medicaments and methods of using the same are disclosed for treating or preventing diseases resulting from undesirable cell adhesion of IL-1 receptor positive cells to biological materials, particular to endothelial cells, or autoimmune related diseases, preferably graft versus host disease, or IL-1 dependent cancers.

This application is a continuation of U.S. Ser. No. 07/791,474 filedNov. 8, 1991, now abandoned, which is a continuation-in-part of U.S.Ser. No. 07/594,827, filed Oct. 9, 1990, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/517,276, filed May 1, 1990, nowabandoned.

FIELD OF THE INVENTION

This invention is in the area of medicinal chemistry, and presentsmedically useful compositions consisting of IL-1 receptor antagoniststhat have prophylactic or therapeutic applications for treating avariety of diseases including endothelial cell associated diseases,autoimmune related diseases, and cancer.

BACKGROUND OF THE INVENTION

Cytokines are small molecular weight proteins that have a myriad ofnormal biological functions as well as being associated with variousdiseases. For example,, the cytokines interleukin-1 (IL-1) and tumornecrosis factor (TNF) have been demonstrated to have multiple biologicalactivities with the two prominent being fever production and leukocyteactivation. Moreover, both cytokines, alone or in combination, cause ashock state in animals that hemodynamically and hematologically ischaracteristic of septic shock in man caused by bacterial infection.TNF, in addition, has recently been shown to be involved in initiatingthe expression of human immunodeficiency virus in human cells that carrylatent virus. Folks et al., 1989, PNAS (USA), 86:2365. TNF and IL-1 alsoplay a role in various autoimmune diseases, particularly arthritis.Duff, et al., 1987, International Conference on Tumor Necrosis Factorand Related Cytotoxins, 175:10.

Endothelial cell injury, or injury to the vascular system, can occur asa result of a number of disease in which there appears to be cytokineinvolvement. For example, ischemia-related injury to either cells,tissues or organs is responsible for many significant clinicaldisorders, including stroke, vascular disease, organ transplantation,and myocardial infarction. Leukocytes, particularly, neutrophils ormonocytes, are thought to be the primary causative agent and have beenshown to cause extensive vascular tissue damage arising as a result ofthe release of oxygen-derived free radicals, as well as proteases andphospholipases from the leukocytes at the site of injury. Harlan, J. M.,1987, Acta. Med. Scand. Suppl., 715:123; Weiss, S., 1989, New England J.of Med., 320:365. Cytokines are thought to be chemotactic agents forleukocytes and may be involved in attracting them to the site of tissueinjury. Additionally, other studies have shown that cytokines areinvolved in causing leukocytes to adhere to the vascular endothelialcell layer which sets the stage for the release of noxious chemicalsthat cause vascular tissue damage.

Further evidence for the role of leukocytes in disease comes fromstudies which have shown that animals depleted of peripheral bloodleukocytes show significantly reduced damage from myocardial ischemiaand reperfusion. Further, reperfusion injury can be minimized by in vivoadministration of monoclonal antibody to an adhesion protein present onleukocytes, termed MAC-1 (discussed more below).

Finally, a rabbit model of hemorrhagic shock and resuscitation revealsthat monoclonal antibodies against the β subunit of MAC-1 exhibited aprotective effect to liver and the asternal intestinal track. Simpson,et al., 1988, J. of Clinical Invest., 81:624; Vedder, N. and Harlan, J.,1988, J. of Clinical Invest., 81:676.

The foregoing studies suggests significant therapeutic value forreagents that block the adhesion of leukocytes in controlling tissue andorgan injury resulting from a number of disease situations includingmyocardial infarction, hemorrhagic shock, and other events that causeischemia that are followed by reestablishing normal circulatory bloodflow.

The initial event that leads to leukocyte, particularly neutrophil,damage of endothelial cells is the adhesion of neutrophils to theendothelial cell surface. In significant part this is mediated bycellular adhesion molecules associated with the neutrophils that causethem to bind to the endothelial cell surface. The neutrophil adhesionmolecules bind to a molecule on the surface of endothelial cells termedICAM-1 (Intercellular Adhesion Molecule 1). ICAM-1 is induced by one ofseveral cytokines, including IL-1 and TNF. To date, a partial list ofthe adhesion molecules that have been identified that are involved inthis reaction are lymphocyte function-associated antigen-1 (LFA-1),macrophage antigen-1 (MAC-1), also termed MO-1, OKM-1 and complementreceptor type-3 (CR-3), and p150,95, also termed complement receptortype-4 (CR-4) and Leu M-5. These molecules collectively have been termedthe LFA-1 family, leukocyte adhesion proteins, leuCAM, and the leukocyteintegrins. All three molecules are α-β heterodimers. The β subunit isidentical in the three molecules, while the α subunit differs.Kurzinger, K., and Springer, T. A., 1982, J. of Biol. Chem., 257:12412;Sanchez-Madrid, F., et al., 1983, J. Exp. Med., 158:1785; Trowbridge, I.S., and Omary, M. B., 1981, PNAS (USA), 78:3039. Studies have shown thecommon β subunit to play the predominant role in the adhesion-relatedfunctions of these molecules. Recently the cDNA clone that encodes the βsubunit of human LFA-1, MAC-1, and p150,95 has been isolated. Kishimoto,T., et al., 1987, Cell, 48:681; and Law, S. K. A. et al., 1987, EMBOJ.,:915-919.

Studies have implicated the leukocyte integrins in cellular adhesionevents. For example, LFA-1 is involved in antigen-dependent andantigen-independent interactions of immune cells. Springer, T. A., etal., 1987, Annual Review Immun., 5:223; Martz, E., 1986, Hum.Immunology, 18:3. Most telling are studies utilizing a monoclonalantibody to LFA-1, which have revealed that binding to LFA-1 bymonoclonal antibody partially or totally inhibits T lymphocyte adherenceto endothelial cells (Mentzer, S. J., et al. 1986, J. of Cell Physiol.,126:285), fibroblasts (Dustin, N. L., et al., 1986, J. of Immun.,137:245), epidermal keratinocytes (Dustin, N. L., et al., 1988, J. ofSubBiology, 107:321), and hepatocytes (Roos, E., and Roossien, F., 1987,J. of SubBiology, 105:553). Additionally, MAC-1 has been shown to beinvolved in macrophage binding to Leishmania Promastigotes, E. coli, andHistoplasma Capsulatum. Mosser, D. and Edelson, P., 1985, J. of Immun.,135:2785; Wright, S. and Jong, M., 1986, J. of Exp Med., 164:1876;Bullock, W. and Wright, S., 1987, J. of Exp. Med., 165:195. Otherstudies have shown that MAC-1 is involved in neutrophil and monocytechemotaxis, as well as adherence to glass and plastic surfaces, and toendothelial and epithelial cell monolayers. p150,95 is reported to besignificantly involved in peripheral blood monocyte adhesion tosubstrates and endothelial cells, phagocytosis of latex particles, andchemotaxis. Keizer, et al., 1987, Eur. J. of Immun., 17:1317; te Velde,A., et al., 1987, Immunology, 61:261. Further, studies using amonoclonal antibody that is directed to p150,95 have shown it to beutilized in conjugate formation by cytotoxic T lymphocytes.

There are two forms of IL-1:IL-1 α and IL-1 β. Although these moleculesshare limited sequence homology they have similar biological activity.Dinarello, C. A., et al., 1986, Journal Clinical Invest., 77:1734. Bothmolecules have molecular weights of about 17.5 kD, and are produced froma precursor molecule with a molecular weight of about 31 kD.

Because IL-1 has pleiotropic biological activities many of whichadversely affect the organism, it would be expected that the moleculemust be tightly regulated if it is not to be injurious. Indeed, thereare several reports of IL-1 inhibitors that regulate the action of IL-1.IL-1 inhibitory activity has been reported in monocyte conditionedmedium, wherein the monocytes are grown on adherent immune complexes.Arend, W. P., et al., 1985, Journal of Immun., 134:3868. Additionally,an inhibitor has been reported to be present in urine. Seckinger, P., etal., 1987, Journal of Immun., 139:1546. Lastly, two protein inhibitors,purified and cloned, that have interleukin-1 receptor antagonistactivity have been reported. Hannum, et al., 1990, Nature, 343:336;Eisenberg, S., et al., 1990, Nature, 343:341; and Haskill, S., et al.,U.S. Ser. No. 517,276, filed May 1, 1990 now abandoned, Carter, D. etal., 1990, Nature, 344:633.

It is thought that the IL-1 inhibitor present in urine, and which hasbeen partially purified and characterized by Seckinger, P. et al., supraand Seckinger, P., et al., 1987, Journal of Immun., 139:1541 is similar,if not identical to the cloned IL-1 receptor antagonist reported byEisenberg, S., et al., supra; and Carter, D., et al., 1990, Nature, 34:633.

As alluded to above, the leukocyte integrins bind to the cell surfaceprotein, ICAM-1, which in turn is induced by one or more cytokines,particularly IL-1 or TNF. Thus, it will be appreciated that an effectivemodality for preventing endothelial cell injury that occurs duringvarious diseases would be to interfere with the induction of ICAM-1 viamolecules that prevent or interfere with the activity of IL-1, TNF orother molecules that induce ICAM-1.

IL-1 also plays a role in various autoimmune or autoimmune relateddiseases (Duff, et al., 1987, International Conference on Tumor NecrosisFactor and Related Cytotoxins, 175:10), particularly graft versus hostrejection involving bone marrow transplants. This is attributable, atleast in part, to IL-1 induction of IL-2 production by T-cells which inturn promotes the growth of additional T-cells. Thus, the graft from adonor contains a significant number of immunocompetent lymphoid cellsthat can mount an effective destructive reaction against host cells.Bone marrow transplants are often employed to treat various malignantdiseases, including leukemia Generally this involves immunologicallycrippling the leukemic patient, and then transplanting bone marrow froma donor. Unless the lymphoid cells in the donor marrow are suppressedthey can react against recipient tissue antigens, often with direconsequences.

A variety of drugs, and antisera to lymphoid cells are used to treatgraft versus host disease. Particularly useful drugs arecorticosteroids, othiopirne, and cyclosporine. In addition, variousmonoclonal antibodies, alone or when coupled to a cytotoxic agent areavailable for ridding the donor marrow of lymphoid cells. P. S. Russellet al., Annual Review Medicine. 35:63 (1984). These drugs, however, havesignificant untoward side effects.

Finally, it has recently been suggested that IL-1 is an autocrine andparacrine growth factor for particular cancers. Tsai, 1987, J. Natl.Cancer Inst., 79:77. Thus, compounds that interfere with the cancergrowth activity of IL-I may be effective chemotherapeutics.

SUMMARY OF THE INVENTION

The invention presented herein describes applications of medicamentsconsisting of IL-1 receptor antagonists for preventing or treatingdiseases, as well as methods for preventing or treating diseases usingIL-1 receptor antagonists in appropriate assay formats.

A second aspect of the invention presents applications of medicamentsconsisting of IL-1 receptor antagonists for preventing or treatingdiseases caused by the undesirable adhesion of leukocytes to endothelialcells, or for preventing or treating autoimmune related diseases,preferably graft versus host disease, or for treating IL-1 dependentcancers.

A third aspect of the invention presents applications of medicamentsconsisting of IL-1 receptor antagonists for preventing or treatingdiseases that result in endothelial cell damage, preferablyischemia-related diseases, including stroke, vascular disease, organtransplantation, and myocardial infarction.

A fourth aspect of the invention is the description of medical uses ofintracellular IL-1 receptor antagonists for preventing or treatingdiseases that result in endothelial cell damage, preferablyischemia-related diseases, including stroke, vascular disease, organtransplantation, and myocardial infarction, or for preventing ortreating autoimmune related diseases, preferably graft versus hostdisease, or for treating IL-1 dependent cancers.

A fifth aspect of the invention is a method of treating or preventingHIV infection in a host consisting of incorporating into host cells anucleotide sequence that encodes IL-1 receptor antagonist activity.

These and other aspects of the invention will be apparent upon a fullconsideration of the invention as presented below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 discloses a DNA sequence (SEQ ID NO: 1) and the correspondingamino acid sequence (SEQ ID NO: 2) of the intracellular IL-1 receptorantagonist encoded thereby.

FIG. 2 shows the HIV infection of transfected CEM cells, (i.e.,transfected human T cell) as measured by reverse transcriptase (RT)activity (CPM) for (1) non-infected CEM cells ("mock"); (2) CEM cellsinfected with the empty plasmid ("pcDNA"); (3) CEM T cells transfectedto express soluble IL-1 receptor antagonist cDNA in pcDNA ("sRA(+)");(4) CEM T cells transfected to express intracellular IL-1 receptorantagonist ("icRA(+)"); and (5) untransfected CEM T cells ("icRA(-)").

Table 1 shows the effect of cellular COS cell lysates prepared from COScells that were mock transfected, transfected with the vector, SRα, withor without the IL-1 receptor antagonist DNA sequence, or containing thesequence in the wrong orientation, or with a vector that encodes IL-2.

Table 2 shows the effect of COS cell lysates on IL-2 production fromLBRM-33 cells when the lysates are prepared from mock transfected cells,cells containing the vector, SRα, that lacks the IL-1 receptorantagonist DNA sequence, the SRα vector that contains the IL-1 receptorantagonist DNA sequence, but in the wrong orientation, and the SRαvector that contains the IL-1 DNA sequence in the proper orientation.

Table 3 shows the effect of COS cell lysates on the inhibition ofprostaglandin production in IL-1 inducible fibroblasts. COS cell lysateswere prepared from COS cells that were mock transfected, transfectedwith the vector, SRα, with or without the IL-1 receptor antagonist DNAsequence, or containing the sequence in the wrong orientation.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein draws on previously published work. Byway of example, such work consists of scientific papers, patents orpending patent applications. All of these publications and applications,cited previously or below, are hereby incorporated by reference relativeto the propositions cited.

The instant invention is composed of several unique methods andcompositions. Each aspect of the invention will now be discussedseparately.

"IL-1 Receptor Antagonist" refers to virtually any molecule thatinterferes with, or prevents the binding of IL-1 to its receptor. By wayof example, and not by way of limitation, such antagonist areexemplified herein by two protein molecules that have been purified andcloned, that have interleukin-I receptor antagonist activity have beenreported. Hannum, et al., 1990, Nature, 343:336; Eisenberg, S., et al.,1990, Nature, 343:341; and Haskill, S., et al., U.S. Ser. No. 517,276,filed May 1, 1990. Other IL-1 Receptor Antagonist would include antibodythat binds to, and interferes with or prevents the binding of IL-1 toits receptor. By antibody is intended any molecule that would have ananti-IL-1 receptor antibody combining site and would include, of course,polyclonal or monoclonal antibody or fragments derived therefrom, andmolecules that have antibody binding activity made by recombinantmethods. Monoclonal antibody to the IL-1 receptor may be produced usingthe general procedures described by Kohler, G. and Milstein, C., 1975,Nature, 256:495, which have been modified over the years as is known inthe art Antibody that binds to the receptor and interferes with IL-1binding are readily identified using competition binding assays known inthe art.

It is important to note that the definition of IL-1 receptor antagonistis not meant to exclude such molecules from having other properties. Forexample, it has been surprisingly discovered that in some instances suchmolecules have agonist activity as well. This is shown by the results inExample 6, wherein the intracellular IL-1 receptor antagonist downregulates CD4 expression on a T-cell line despite such cells notexpressing detectible levels of IL-1.

"Cells" or "recombinant host" or "host cells" are often usedinterchangeably as will be clear from the context. These terms includethe immediate subject cell, and, of course, the progeny thereof. It isunderstood that not all progeny are exactly identical to the parentalcell, due to chance mutations or differences in environment.

As used herein the term "transformed" in describing host cell culturesdenotes a cell that has been genetically engineered to produce aheterologous protein that possesses the activity of the native protein.Examples of transformed cells are described in the examples of thisapplication. Bacteria are preferred microorganisms for producing theprotein. Synthetic protein may also be made by suitably transformedyeast and mammalian host cells.

"Control sequences" refers to DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences which are suitable for procaryotes, for example,include a promoter, optionally an operator sequence, a ribosome bindingsite, and possibly, other as yet poorly understood, sequences.Eucaryotic cells are known to utilize promoters, polyadenylationsignals, and enhancers.

"Expression system" refers to DNA sequences containing a desired codingsequence and control sequences in operable linkage, so that hoststransformed with these sequences are capable of producing the encodedproteins. In order to effect transformation, the expression system maybe included on a vector; however, the relevant DNA may then also beintegrated into the host chromosome.

As used herein, the term "pharmaceutically acceptable" refers to acarrier medium which does not interfere with the effectiveness of thebiological activity of the active ingredients and which is not toxic tothe hosts to which it is administered. The administration(s) may takeplace by any suitable technique, including subcutaneous and parenteraladministration, preferably parenteral. Examples of parenteraladministration include intravenous, intraarterial, intramuscular, andintraperitoneal, with intravenous being preferred.

As used herein, the term "prophylactic or therapeutic" treatment refersto administration to the host of the cytokine inhibitor either before orafter infection or cancer detection. If the cytokine inhibitor isadministered prior to exposure to the infecting agent, the treatment isprophylactic (i.e., it protects the host against infection), whereas ifadministered after infection or initiation of cancer, the treatment istherapeutic (i.e., it combats the existing infection or cancer).

I. IL-1 Receptor Antagonists

A. Isolated/Cloned Protein Antagonist:

FIG. 1 shows the amino acid sequence of the intracellular form of theIL-1 receptor antagonist (SEQ ID NO:2). The cDNA sequence that encodesthe molecule has been described in U.S. patent Ser. No. 517,276, filedMay 1, 1990, and in Haskill, S. et al., PNAS, in press. The cDNAsequence (SEQ ID NO:1) is on deposit with the American Type CultureCollection and the Cetus Microbial Culture Collection and has ATCCAccession No. 68449 and CMCC No. 3827. A secreted form of theintracellular IL-1 receptor antagonist is described by Hannum, et al.,1990, Nature, 343:336; Eisenberg, S., et al., 1990, Nature, 343:341.Carter et al., supra.

The IL-1 receptor antagonist cDNA sequence (SEQ ID NO:1) can beexpressed in a number of expression systems, and in a wide variety ofcell types. Procaryotes most frequently are represented by variousstrains of E. coli. However, other microbial strains may also be used,such as bacilli, for example, Bacillus subtilis, various species ofPseudomonas, or other bacterial strains. In such procaryotic systems,plasmid vectors which contain replication sites and control sequencesderived from a species compatible with the host are used. For example,E. coli is typically transformed using derivatives of pBR322, a plasmidderived from an E. coli species by Bolivar et al., 1977, Gene 2:95.pBR322 contains genes for ampicillin and tetracycline resistance, andthus provides additional markers which can be either retained ordestroyed in constructing the desired vector. Commonly used procaryoticcontrol sequences, which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta-lactamase (penicillinase) and lactose (lac) promoter systems(Chang et al., 1977, Nature 198:1056), the tryptophan (tip) promotersystem (Goeddel E al., 1980, Nucleic Acids Res. 8:4057) and the lambdaderived P_(L) promoter (Shimatake et al, 1981, Nature 292:128), andN-gene ribosome binding site, which has been made useful as a portablecontrol cassette, U.S. Pat. No. 4,711,845, issued Dec. 8, 1987, andincorporated herein by reference in its entirety, which comprises afirst DNA sequence that is the P_(L) promoter operably linked to asecond DNA sequence corresponding to the N_(RBS) upstream of a third DNAsequence having at least one restriction site that permits cleavagewithin 6 bp 3' of the N_(RBS) sequence. U.S. Pat. No. 4,666,848, issuedMay 19, 1987, and incorporated herein by reference in its entiretydiscloses additional vectors with enhanced expression capabilities. Alsouseful is the phosphatase A (phoA) system described by Chang et al., inEuropean Patent Publication No. 196,864, published Oct. 8, 1986,incorporated herein by reference. However, any available promoter systemcompatible with procaryotes can be used.

In addition to bacteria, eucaryotic microbes, such as yeast, may also beused as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker'syeast, are most used, although a number of other strains are commonlyavailable. While vectors employing the 2 micron origin of replicationare illustrated (Broach, 1983, Meth. Enz., 101:307; U.S. Pat. No.4,803,164, incorporated herein by reference in its entirety), otherplasmid vectors suitable for yeast expression are known (see, forexample, Stinchcomb et al., 1979, Nature 282:39, Tschempe et al., 1980,Gene 10:157 and Clarke et al., 1983, Meth. Enz. 101:300). Controlsequences for yeast vectors include promoters for the synthesis ofglycolytic enzymes (Hess et al., 1968, J. Adv. Enzyme. Req. 7:149;Holland et al., 1978, Biochemistry 17:4900).

Additional promoters useful in yeast host microorganisms and known inthe art include the promoter for 3-phosphoglycerate kinase (Hitzeman etal., 1980, J. Biol. Chem. 255: 2073), and those for other glycolyticenzymes, such as glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Other promoters,which have the additional advantage of transcription controlled bygrowth conditions, are the promoter regions for alcohol dehydrogenase 2,isocytochrome C, acid phosphatase, degradative enzymes associated withnitrogen metabolism, and enzymes responsible for maltose and galactoseutilization (Holland, supra ).

It is also believed that terminator sequences are desirable at the 3'end of the coding sequences. Such terminators are found in the 3'untranslated region following the coding sequences in yeast-derivedgenes. Many of the vectors illustrated contain control sequences derivedfrom the enolase gene containing plasmid peno46 (Holland et al., 1981,J. Biol. Chem. 256:1385) or the LEU2 gene obtained form YEp13 (Broach etal., 1978, Gene 8:121); however, any vector containing a yeastcompatible promoter, origin of replication and other control sequencesis suitable.

It is also, of course, possible to express genes encoding proteins ineucaryotic host cell cultures derived from multicellular organisms. See,for example, Tissue Culture Academic Press, Cruz and Patterson, editors(1973). Useful host cell lines include murine myelomas N51, VERO andHeLa cells, and Chinese hamster ovary (CHO) cells. Expression vectorsfor such cells ordinarily include promoters and control sequencescompatible with mammalian cells such as, for example, the commonly usedearly and late promoters from Simian Virus 40 (SV 40) (Fiers et al.,1978, Nature, 273:113) viral promoters such as those derived frompolyoma, Adenovirus 2, bovine papilloma virus, or avian sarcoma viruses,or immunoglobulin promoters and heat shock promoters. A system forexpressing DNA in mammalian systems using the BPV as a vector isdisclosed in U.S. Pat. No. 4,419,446, incorporated herein by referencein its entirety. A modification of this system is described in U.S. Pat.No. 4,601,978, incorporated herein by reference in its entirety. Generalaspects of mammalian cell host system transformations have beendescribed by Axel in U.S. Pat. No. 4,399,216, issued Aug. 16, 1983. Alsouseful is gene amplification in eucaryotic cells as described by Ringoldin U.S. Pat. No. 4,656,134, issued Apr. 7, 1987, incorporated herein byreference in its entirety. It now appears also that "enhancer" regionsare important in optimizing expression; these are, generally, sequencesfound upstream of the promoter region. Origins of replication may beobtained, if needed, from viral sources. However, integration into thechromosome is a common mechanism for DNA replication in eucaryotes.

Plant cells are also now available as hosts, and control sequencecompatible with plant cells such as the nopaline synthase promoter andpolyadenylation signal sequences (Depicker et al., 1982, J. Mol. Appl.Gen., 1:561) are available. Additionally, methods and vectors fortransformation of plant cells have been disclosed in PCT Publication No.WO 85/04899, published Nov. 7, 1985, and incorporated herein byreference in its entirety.

It will be appreciated by those skilled in the art that although theprecise chemical structure of the IL-1 receptor antagonist is shown inFIG. 1, and conservative amino acid changes or deletions or in the caseof antibody antagonists are readily determined using known sequencingmethods, the primary amino acid sequence of the protein may be augmentedby derivatization using sugar moieties (glycosylation) or by othersupplementary molecules such as lipids, phosphate, acetyl groups and thelike, as well as by conjugation with saccharides, polyethylene glycols(PEGs) and polyoxyethylene glycols (POGs). Such modifications areincluded in the definition of peptide herein so long as the activity ofthe peptide, as defined above, is not destroyed. It is expected, ofcourse, that such modifications may quantitatively or qualitativelyaffect the activity, either by enhancing or diminishing the activity ofthe protein in the various assays.

B. Antibody IL-1 Receptor Antagonists:

Procedures for producing antibody, polyclonal or monoclonal, are wellknown in the art and may be adapted to produce IL-1 receptor antibodythat binds to the receptor and prevents or interferes with IL-1 binding.The methods for generating monoclonal antibody are described by Kohler,G. and Milstein, C., 1975, Nature, 256:495. These initial studiesinvolved fusing murine lymphocytes and drug selectable plasmacytomas toproduce hybridomas. Subsequently, the technique has been applied toproduce hybrid cell lines that secrete human monoclonal antibodies. Thelatter procedures are generally described in Abrams, P., 1986, Methodsin Enzymology, 121:107, but other modifications are known to thoseskilled in the art. Regardless of whether murine or human antibody isproduced, the antibody secreting cells are combined with the fusionpartner and the cells fused with a suitable fusing agent, preferablypolyethylene glycol, and more preferably polyethylene glycol 1000. Thelatter is added to a cell pellet containing the antibody secreting cellsand the fusion partner in small amounts over a short period of timeaccompanied with gentle agitation. After the addition of the fusingagent, the cell mixture is washed to remove the fusing agent and anycellular debris, and the cell mixture consisting of fused and unfusedcells seeded into appropriate cell culture chambers containing selectivegrowth media. After a period of several weeks, hybrid cells areapparent, and may be identified as to antibody production and subclonedto ensure the availability of a stable hybrid cell line.

The preferred antibody is human monoclonal antibody which can beprepared from lymphocytes sensitized with IL-1 receptor, or with cellsthat bear the same, either in vivo or in vitro by immortalization ofantibody-producing hybrid cell lines, thereby making available apermanent source of the desired antibody. In vivo immunizationtechniques are well known in the art, while in vitro techniques aregenerally described by Luben, R. and Mohler, M., 1980, MolecularImmunology, 17:635, Reading, C. Methods in Enzymology, 121 (PartOne):18, or Voss, B., 1986, Methods in Enzymology, 121:27. A number ofin vitro immunization systems have been shown to be effective forsensitizing human B-cells. Reading, C., 1982, J. of Immun. Methods,53:261.

Sensitized lymphocytes can be immortalized by viral transformation. Thepreferred viral transformation technique for human lymphocytes involvesthe use of Epstein-barr virus. The virus is capable of transforminghuman B-cells, and has been used to generate human monoclonalantibodies. Crawford, D. et al., 1983, J. of General Virology, 64:697;Kozbor, V. and Roder, J., 1983, J. Immun. Today, 4:72.

Another procedure whereby sensitized lymphocytes may be immortalizedconsist of a combination of the above two techniques, that is viraltransformation and cell fusion. The preferred combination consist oftransforming antibody secreting cells with Epstein-barr virus, andsubsequently fusing the transformed cells to a suitable fusion partner.The fusion partner may be a mouse myeloma cell line, a heteromyelomaline, or a human myeloma line, or other immortalized cell line. PCTPatent Application No. 81/00957; Schlom et al., 1980, PNAS (USA),77:6841; Croce et al., 1980, Nature, 288:488. The preferred fusionpartner is a mouse-human hetero-hybrid, and more preferred is the cellline designated F3B6. This cell line is on deposit with the AmericanType Culture Collection, Accession No. HB8785. It was deposited Apr. 18,1985. The procedures for generating F3B6 are described in EuropeanPatent Application, Publication No. 174,204.

Techniques applicable to the use of Epstein-Barr virus transformationand the production of immortal antibody secreting cell lines arepresented by Roder, J. et al., 1986, Methods in Enzymology, 121:140.Basically, the procedure consist of isolating Epstein-Barr virus from asuitable source, generally an infected cell line, and exposing thetarget antibody secreting cells to supernatants containing the virus.The cells are washed, and cultured in an appropriate cell culturemedium. Subsequently, virally transformed cells present in the cellculture can be identified by the presence of the Epstein-Barr viralnuclear antigen, and transformed antibody secreting cells can beidentified using standard methods known in the art.

The hybridoma cells may be grown in appropriate cell culture media, andthe supernatant screened for the presence of monoclonal antibody thatprevents or interferes with binding of IL-1 to its receptor using, forexample, an enzyme linked immunosorbent assay (ELISA). The latter isdescribed by Engvall, E., 1977, Med. Biol., 55:193. Basically, theprocedure consist of coating flat-bottom 96 well microtitre plates withthe appropriate source of IL-1 receptor antigen, preferably eitherpurified IL-1 receptor or cells that carry the receptor, reacting theantigen with cell culture supernatant, removing the supernatant and thenrevealing the presence of antibody using a suitable second antibody thathas been labelled with a reporter group. It is well known in the artthat the reporter group is preferably a radioactive tracer or afluorescent molecule. Procedures for isolating IL-1 receptor aredescribed in European patent application, Publication No. 318,296, toDower, S. K. et al., published May 31, 1989.

Those hybridomas that initially give a positive signal and thereforesecrete antibody that bindings to the receptor are retested in thepresence of IL-1 to determine if the antibody has IL-1 receptorantagonist activity. This assay is preferably conducted using labelledIL-1 and measuring a reduction in IL-1 binding to the receptor in thepresence of antibody.

It will be apparent to those skilled in the art that while the preferredembodiment of the instant invention is anti-IL-1 receptor monoclonalantibody, singly or in combination, that the antibody(s) may be alteredand still maintain biological activity. Thus, encompassed within thescope of the invention is antibody modified by reduction to various sizefragments, such as F(ab')₂, Fab, Fv, or the like. Also, the hybrid celllines that produce the antibody may be considered to be a source of theDNA that encodes the desired antibody, which may be isolated andtransferred to cells by known genetic techniques to produce geneticallyengineered antibody. An example of the latter would be the production ofsingle chain antibody having the antibody combining site of thehybridomas described herein. Single chain antibody is described in U.S.Pat. No. 4,704,692, or in U.S. Pat. No. 4,946,778. A second example ofgenetically engineered antibody is recombinant, or chimeric antibody.Methods for producing recombinant antibody are shown in U.S. Pat. No.4,816,567, inventor Cabilly, et al.; Japanese patent application, SerialNo. 84169370, filed Aug. 15, 1984; U.S. patent application, Serial No.644,473, filed Aug. 27, 1984; British patent application 8422238, filedon Sep. 3, 1984; Japanese patent application, No. 85239543, filed Oct.28, 1985; U.S. patent application, Ser. No. 793,980 on Nov. 1, 1985 nowabandoned; U.S. patent application, Ser. No. 77,528, filed Jul. 24, 1987now abandoned. Also, British patent application, No. 867679, filed Mar.27, 1986 describes methods for producing an altered antibody in which atleast parts of the complementary determining regions (CDRs) in the lightor heavy chain variable domains have been replaced by analogous parts ofCDRs from an antibody of different specificity. Using the proceduresdescribed therein it is feasible to construct recombinant antibodyhaving the CDR region of one species grafted onto antibody from a secondspecies that has its CDR region replaced. The preferred embodiment inthis instance is a murine anti-IL-1receptor antibody CDR region thatreplaces the CDR region of human antibody. Hybrid cells produced byfusing antibody secreting cells and an appropriate fusion partner, orEpstein-Barr virus transformed cells that produce the desired antibodycan be identified using convenient immunochemical screening techniques.

Regardless of whether the antibody being screened is monoclonal,recombinant, single chain etc., the assay described above may beemployed to identify IL-1 receptor antagonists.

C. Formulation of IL-1 Receptor Antagonists:

It will be appreciated by those skilled in the art that the IL-1receptor antagonists described herein can be administered to mammals,including humans, either alone or in combination with otheranti-inflammatory agents, or they may be combined with variouspharmaceutically acceptable diluents or carriers. Such are widely knownto those skilled in the art and are formulated according to standardpharmaceutical practices.

Exemplary diluents include physiologic saline, or buffered saline, aswell as Ringer's and dextrose injection fluid, and dextrose saline andlactated Ringer's injection or diluent solutions containing additionaltherapeutic agents, preferably antibiotics or antibodies known to beefficacious in the treatment of inflammatory conditions.

II. Leukocyte Preparation/Labelling

Leukocyte adherence can be measured using several assays known in theart, and the preferred assay is described by Charo, et al., 1985, Blood,65:473. Briefly, the assay consists of labelling leukocytes with anappropriate label, incubating the leukocytes with endothelial cells anddetermining the number of leukocytes that adhere. Preferably the cellsare labelled with a gamma ray emitting isotope and the preferred labelsare ¹¹¹ Indium-oxide or ⁵¹ chromium.

Leukocytes may be isolated from human donors using standard techniques.This generally consists of isolating blood in a physiologically balancedsalt solution containing an appropriate anticoagulant, and separatingthe leukocytes by an appropriate separation step, preferably onFicoll-Hypaque gradients. Contaminating erythrocytes can be removed byhypotonic lysis. The resulting leukocytes are suspended in aphysiologically buffered solution, pH 7.4. The preferred physiologicalbuffered solution is Hank's balanced salt solution that is calcium andmagnesium free.

The isolated leukocytes can then be labelled by incubating them for anappropriate time, generally 15 minutes, with the desired radioisotope ata predetermined concentration. The radiolabelled cells are washed toremove unincorporated label, and then suspended in an appropriatesolution to perform the adhesion assay described below.

III. Endothelial Cell Preparation/Culture

Endothelial cells can be prepared from a number of sources and byseveral techniques known in the art. Preferably they are obtained fromhuman umbilical veins using the procedure of Charo et al., above.Generally, endothelial cells are isolated by enzymatic digestion of theumbilical veins using, preferably, collagenase as described by Jaffe, E.A., et al., 1973, J. of Clin. Invest, 52:2745. The cells are grown on anappropriate tissue culture substratum, preferably gelatine-coatedsurfaces.

The endothelial cells may be grown in a variety of tissue culture mediacontaining appropriate supplements such as an appropriate concentrationof fetal calf serum, and other supplements/additives routinely utilizedby those skilled in this art that are recognized as being favorable forendothelial cells. The endothelial cells may be passaged with a dilutesolution of an appropriate protease, and if desired a metal ionchelator. Preferably a solution consisting of 0.05 to 0.25% trypsin and0.02% EDTA is used. To ensure that the cells are indeed endothelialcells, they are tested by immunofluorescence for Factor VIII antigen, aknown endothelial cell marker.

IV. Leukocyte/Endothelial Cell Adhesion Assay

Leukocyte adherence to endothelial cell monolayers may be determined asfollows. Early passage endothelial cells, generally not beyond the fifthpassage, are cultured on an appropriate substratum and in a suitablecell culture medium. The culture substratum is preferably pre-coatedwith an appropriate substance that enhances the adherence of theendothelial cells. Several such substances are known includingfibronectin, poly-L-lysine, gelatin and laminin. Fibronectin ispreferred. An appropriate culture substratum is a 96-well micro titerplate, and a suitable medium is Medium 199 containing fetal calf serumand other supplements known to be beneficial for the growth andmaintenance of endothelial cells that are well known to those skilled inthe art. The endothelial cell monolayer is washed with a physiologicallybalanced salt solution containing a reduced amount of fetal calf serum,preferably 1%. The preferred solution is RPMI supplemented with 1% fetalcalf serum. Prior to the adhesion step, endothelial cells and/orleukocytes are incubated with IL-1 to induce membrane adhesionmolecules. Preferably 10 U/ml IL-1 β is added to the endothelial cellsfor 4 hours.

The endothelial cell monolayer containing added leukocytes preferably¹¹¹ Indium monocytes are incubated for a time sufficient to permitmaximum adherence of the leukocytes, and preferably this is conducted at37° C. for 30 minutes in an appropriate cell culture atmosphere.Generally this would consist of incubating the cells for the assayperiod in 5% CO₂, 95% air, and 95% humidity. Next, non-adherentleukocytes are removed by any number of techniques known in the art, andthe number of leukocytes adherent to the endothelial cell monolayersdetermined by measuring the amount of radioisotope associated with theendothelial cell monolayer. Controls are run that take into accountbasal binding, i.e., binding to endothelial cells not activated withIL-1.

In a typical experiment run in quadruplicate, the assay is highlyreliable, giving standard deviations less than 10%, and usually lessthan 5%, of mean values. Typically the results are expressed as thepercent of leukocytes added to the endothelial cells that remainadherent after non-adherent cells have been removed.

Using the above assay, typically the IL-1 receptor antagonist is addedover a range of concentrations and preferably at 10⁻⁴ M. This volume isthen suitably diluted in an appropriate medium, preferably RPMIcontaining 1% fetal calf serum to give the desired final concentrationto be tested.

The endothelial cells were activated with 10 U/ml of IL-1 β (GenzymeCorp.) for at least 4 hours in RPMI with 1% fetal calf serum prior tothe addition of the leukocytes. IL-1 causes the induction of ICAMexpression on endothelial surfaces which is a receptor for leukocyteintegrin binding.

V. Determination of the Effectiveness of an IL-1 Receptor Antagonist inGraft Versus Host Disease Using Primary Mixed Lymphocyte andPhytohemagglutinin Assays

The mixed lymphocyte response (MLR) and phytohemagglutinin A (PHA)assays are valuable for identifying immune suppressive molecules invitro that are useful for treating graft versus host disease. Theresults obtained from these assays are generally predictive of their invivo effectiveness.

The in vitro mixed lymphocyte assay is presently employed in theclinical setting as an indicator of histocompatibility, and is premisedon the transformation of resting genetically dissimilar lymphocytes intocells which synthesize DNA and undergo proliferation. It has beendemonstrated that incompatibility at the major histocompatibilitycomplex is mainly responsible for this phenomenon.

A second assay widely used to study immune responsiveness is mitogenicstimulation of lymphocytes with mitogenic substances of plant origin.The most widely used plant molecule is PHA. Although PHA stimulates DNAsynthesis non-specifically in a large number of lymphocytes, unlike trueantigenic stimulation which causes mitogenesis of sub-populations oflymphocytes, the susceptibility of a patient's lymphocytes to PHAstimulation has been shown to correlate with the overall immuneresponsiveness of the patient.

Thus, it will be appreciated as to both the mixed lymphocyte and PHAassay that they are valuable for identifying immune suppressivemolecules in vitro, and that the results obtained therefrom aregenerally predictive of their in vivo effectiveness.

In addition to the above immunosuppressive assays, a secondary mixedlymphocyte reaction assay may also be used. The secondary mixedlymphocyte assays differs from the primary mixed lymphocyte reactionassays in that they employ many more primed responder cells that areresponsive to the primary stimulating cells. The presence of suchresponsive cells is a reflection of immunological memory in an ongoingimmunological response. The protocol for carrying out a secondary mixedlymphocyte assay involves performing a primary lymphocyte assay asdescribed above, and recovering viable cells about 9-10 days after theprimary mixed lymphocyte reaction exhibits little or no cellproliferation. Generally between 10% to 50% of the original input cellsare recovered in viable condition. These cells are then used in thesecondary mixed lymphocyte reaction.

The procedure for carrying out a secondary mixed lymphocyte reaction isdescribed by T. Meoen, Immunological Methods, Eds I. Lefkoivits and B.Pernis, Economic Press, New York (1979). It will be appreciated thatdescribed therein is a method for carrying out secondary lymphocytereactions using mouse cells, however such methods are generallyapplicable to performing secondary mixed lymphocyte reactions usinghuman cells with modifications that are well known to those skilled inthe art.

Using one or all of the above assays, the immune suppressiveeffectiveness of IL-1 receptor antagonists can be determined, and thustheir use in treating graft versus host disease defined. The assays canbe performed as is known in the art, and with or without added IL-1receptor antagonists. The antagonists would be added to the assayreaction mixture at various concentrations to ascertain theconcentration that is optimally effective.

Having described what the applicants believe their invention to be, thefollowing examples are presented to illustrate the invention, and arenot to be construed as limiting the scope of the invention. For example,variation in the source, type, or method of producing antibodies;different labels and/or signals; test supports of different materialsand configurations; different immobilization methods may be employedwithout departing from the scope of the present invention.

Example 1 Effect of IL-1 Receptor Antagonist on Monocyte Adhesion toEndothelial Cell Monolayers

COS cell lysates containing IL-1 receptor antagonist were tested fortheir capacity to interfere with, or block adhesion of monocyte to humanendothelial cell monolayers. A variety of controls were also runincluding a buffer control or performing the assay using lysatesprepared from cells transfected with the vector, SRα, without the IL-1receptor antagonist DNA sequence or with the sequence in the wrongorientation. Also, the effects of IFN, and IL-2 were tested. COS cellswere cultured in MEM (minimal essential medium) with high glucose, +10%FBS.

COS cell lysates were prepared from COS-7 cells that were transfectedwith plasmid DNA containing the PstI fragment, which contains all of the5' untranslated, the complete open reading frame, and 60 based pairs ofthe 3' untranslated region. Transfection was achieved using theDEAE-dextran/chloroquine technique as described by Wong, G. G. et al.,1985, Science, 228:810. Three days subsequent to the transfection, thesupernatant and cells were freeze thawed to obtain a culture lysate.Suitable dilutions of the lysate were employed in the experimentsdescribed below.

The endothelial cells were isolated from human umbilical cords by mildcollagenase digestion. Collagenase was obtained from WorthingtonCorporation, Freehole, N.J., and the general procedure is described byJaffe, E. A., et al., 1973, J. of Clin. Invest., 52:2745. The cellsobtained from collagenase digestion were grown on gelatin-coated flasksin cell culture medium consisting of medium 199 (Gibco,Grand Island,N.Y.) buffered with 25 mM Hepes. The media was supplemented with 20%fetal calf serum. The media also contained 60 μg/ml sodium heparin(Sigma Corporation, St. Louis, Mo.), 2 mM L-glutamine and 50 μg/ml ofbovine hypothalamus extract. The bovine tissue was obtained from PelFreeze, Rogers, AK. The hypothalamus extract serves as a source ofendothelial cell growth factor. The pH of the cell culture media was7.4.

After the endothelial cells reach confluency, they are passaged with0.25% trypsin containing 0.02% EDTA, and subsequent subculturing wasperformed using the same solution. The cells were exposed to thismixture in Hank's balanced salt solution at room temperature for about 1minute.

Finally, approximately 2×10⁴ cells/well were seeded in microtiterplates. The endothelial cell nature of the cells was confirmed both bytheir cobblestone morphology at confluency, and the fact that theystained positive for Factor VIII antigen by indirect immunofluorescence.The latter procedure is well known in the art, and is described byJaffe, E. A., 1973, J. Clin. Invest. 52:2745.

Monolayers of endothelial cells, prior to the fifth passage, wereestablished on polystyrene, 96-well flat bottom micro titer plates(Coming Corporation) in Medium 199 containing 20% fetal calf serum 25 mMhepes, pH 7.4, and the other supplements described above. The surfacesof the micro titer plates were incubated with 6.4 μg/ml human plasmafibronectin for 30 minutes at 25° C. prior to plating the endothelialcells. The solution of fibronectin was removed before addition ofendothelial cells.

The endothelial cell cultures were used when they were confluent. Theendothelial cell monolayers were washed with RPMI plus 1% fetal calfserum and incubated with 10 U/ml of IL-1 β to activate the endothelialcells, and 1:10 dilutions of COS cell lysates. Next, labelled monocytesat a final concentration of 5×10⁵ cells per well were added and allowedto settle for 30 minutes onto the endothelial cell monolayers.

Human monocytes were obtained from venus blood from several healthyadult volunteers using an anti-coagulant (10% heparin) followed bycentrifugation of the blood on Ficoll-Hypaque gradients. Contaminatingerythrocytes were removed by hypotonic lysis. The remaining cellpopulation consisted of 95 to 98% polymorphonuclear leukocytes, andthese cells were suspended at a concentration of 50×10⁶ cells per ml inHank's balanced salt solution, pH 7.4.

The monocytes were labelled with ¹¹¹ Indium-oxide (100 μCi/10⁸ PMNs) (10mCi/mml, Amersham Corp.). Labelling occurred at room temperature inHank's solution for 15 minutes, after which the labelled cells wereisolated by centrifugation for 5 minutes, and to remove residualunincorporated label, washed twice with Hank's balanced salt solution,and then suspended in RPMI supplemented with 1% fetal calf serum.

As mentioned above, 5×10⁵ of the labelled monocytes were added per wellin 96-well micro titer plates. Incubations were conducted for 30 minutesat 37° C., in a tissue culture incubator in an atmosphere of 5% CO₂, 95%air.

After the 30 minute incubation period, during which the monocytes adhereto the endothelial cell monolayer, the micro titer plates were filledand sealed with adherent transparent plastic (Dynatech, Inc., Alexander,Va.), inverted and centrifuged using a micro plate carrier, obtainablefrom Beckman Instruments Corp. Centrifugation was at 75×g for 5 minutesat room temperature. This effectively removed nonadherent PMNs from theendothelial cell monolayers. Next, the plates were blotted dry and thenumber of monocytes that remained adherent to the endothelial cellmonolayers was determined using a gamma counter. The results are shownin Table 2, and they are expressed as the percent of monocytes thatremained adherent to the endothelial cell monolayers. The experimentswere conducted using monocytes isolated from two individuals, donor 1and donor 2.

                  TABLE 1    ______________________________________    Inhibition of Binding of Human Peripheral Blood Monocytes    to IL-1 Stimulated Endothelial Cells             Donor 1      Donor 2    COS Lysate % Binding                        % Change  % Binding                                         % Change    ______________________________________    Mock       14.9     7         17.3   5    SRα  14.1     0         14.4   -15    IL-2       14.8     6         17.2   4    icIL-1ra   14.4     3         15.7   -6    (anti-sense)    icIL-1ra   6.9      -59       6.8    -66    (sense)    ______________________________________

The data shown in Table 1 are instructive in several aspects. First,neither COS cell lysates prepared from mock transfected cells, norlysates prepared from the SRα vector lacking the IL-1 receptorantagonist DNA coding sequence cause a significant or reproducibledecrease in monocyte adhesion to the endothelial cell monolayer.Similarly, neither COS cell lysates prepared from cells transfected withIL-2 vector nor plasmid containing IL-1 receptor antagonist DNA in theanti-sense orientation effect the adhesion of monocytes. In contrast,however, lysates prepared from COS cells transfected with icIL-1ra senseDNA reduce the number of monocytes that adhere to the endothelial celllayer by greater than 50% from both donors.

These results convincingly establish that an IL-1 receptor antagonistcan reduce the adhesion of monocytes to endothelial cells and thus havepositive medical applications as presented in Examples 3 and 4, below.

Example 2 Effect of IL-1 Receptor Antagonist on IL-1 Dependent IL-2Production

To further determine the effectiveness of the IL-1 receptor antagonistto compete with IL-1 for binding to the IL-1 receptor, the capacity ofthe antagonist to inhibit IL-1 induced production of IL-2 from LBRM-331A5 cells was determined LBRM-33 1A5 cells are known to produce IL-2 inresponse to IL-1. LBRM-33 is described by Gillis, S. et al., 1981, PNAS,78:1133 and is on deposit with the ATCC with Accession No. CRL 8079.IL-2 levels were measured using a cellular assay involving a cell linethat requires IL-2 for survival. The cell line employed was HT-2, andthe levels of IL-2 were determined by ³ H-thymidine incorporation afteran 18-24 hour period. HT-2 cells are a mouse IL-2 dependent cell linewhich die in the absence of IL-2. The assay is described by Gillis etal., 1978, J. of Immunol., 120:2027.

LBRM-33 cells were induced with 0.08 U/ml IL-1 β, obtained from CistronBiotechnology, Pine Brook, N.J., plus 2.5 μg/ml PHA, obtained from SigmaChemical Corporation, St. Louis, Mo., and various dilutions of the COScell lysates. After 24 hours LBRM-33 cell culture supernatants weretested for IL-2 using the HT-2 cell assay as described by Gillis, etal., above.

                  TABLE 2    ______________________________________    Inhibition of IL-1 Induced IL-2 Production by LBRM-33 Cells               % Control HT-2 Proliferation               Dilution:    COS Lysate   1:8     1:16   1:32   1:64 1:128    ______________________________________    Mock         42      70     86     91   86    SRα    43      71     88     90   106    IL-2         448     456    460    488  373    icIL-1ra (anti-sense)                 38      62     86     85   90    icIL-1ra (sense)                 6       21     46     57   69    ______________________________________

It is apparent based on the results shown in Table 2 that lysatesprepared from COS cells transfected with icIL-1ra sense DNA inhibit theproduction of IL-2 from LBRM-33 cells. In contrast, COS cell lysatesprepared from mock transfected cells, or cells infected with the vectorSR α that did not contain the intracellular IL-1 receptor antagonist DNAcoding sequence show no inhibitory effect. Two additional lysatecontrols were run. COS cells transfected with SR α encoding IL-2 andlysates prepared from cells transfected with plasmid containing IL-1receptor antagonist DNA in the anti-sense orientation. Neither controlcaused a significant inhibition of IL-2 production. Note that lysatesprepared from COS cells transfected with SR α encoding IL-2 exhibitsignificant IL-2 levels in the assay. This is because IL-2 is carriedover from the IL-2 transfection, and thus produces an apparent positivesignal in the HF-2 assay.

The above results clearly establish that the intracellular form of theIL-1 receptor antagonist effectively blocks the IL-1 induced productionof IL-2.

Example 3 Inhibition of Prostaglandin Production

The effect of the intracellular IL-1 receptor antagonist onprostaglandin production by human fibroblasts was determined. NHDF-194human fibroblasts were obtained from Clonetics Corporation, San Diego,Calif., and cultured using standard methods. Prostaglandin, specificallyprostaglandin E₂, was assayed by RIA using a commercial kit availablefrom Dupont Corporation, NEK-020. The results are shown in Table 3.

COS cell lysates were prepared from COS cells that were transfected withthe vector, SR-α, with the IL-1 receptor antagonist DNA sequence, orcontaining the sequence in the wrong orientation. Inhibitor and IL-1were added to the appropriate lysate at a 150 U/ml (10⁶ U/mg, CistronCorporation) and incubated with 2×10⁴ fibroblasts for 18 hours, afterwhich cultures supernatants were collected and assayed for prostaglandinE₂. Table 3 shows that fibroblasts not exposed to IL-1 do not exhibitelevated levels of prostaglandin, whereas those that were exposed toIL-1 do produce prostaglandin in the range of about 1000 pg/well (0.2ml). Inhibition of prostaglandin production was observed only with COScell lysates prepared from COS cells transfected with the intracellularIL-1 receptor antagonist oriented in the correct orientation. Theinhibited level observed is about an order of magnitude below either ofthe controls.

                  TABLE 3    ______________________________________    IclL-1ra* Blocks IL-1 Induced Fibroblast Prostaglandin Production                     PGE.sub.2    Sample             No IL-1 150 U/ml IL-1    ______________________________________    Control                47-51   790-1072    iclL-1ra*    Correct orientation                  1:4      58      88                  1:8      48      114                  1:16     53      157    Wrong Orientation                  1:4      89      1700                  1:8      62      1120                  1:16     50      990    ______________________________________     *Intracellular IL1 Receptor Antagonist

Example 4 Prophylactic Use of the Intracellular IL-1 Receptor AntagonistFor Reducing Ischemic Injury

The intracellular IL-1 receptor antagonist can be used to reduce orprevent ischemic injury as exemplified by the following example. Anexperimental system with which to test the effectiveness of theintracellular IL-1 receptor antagonist is a rat ligation model. Briefly,this would consist of using rats that weight about 300 grams,anesthetizing them with a suitable anesthetic such as pentabarbial andperforming a thoracectomy. Next, the left anterior descending coronaryartery would be ligated at its origin, and the ligation maintained forabout 1 hour. This effectively reduces or eliminates blood flow thussetting the stage for ischemic injury to the artery. Subsequently, theligation would be released to allow reperfusion, and the reperfusionpermitted to take place for 30 minutes to 1 hour. For the experimentalgroup of animals, reperfusion would be accompanied by administering intothe femoral vein appropriate amounts of the intracellular IL-1 receptorantagonist, and this would range from 50 μg/-1000 μg/per kilogram ofbody weight. The control group would receive a saline solution lackingthe receptor antagonist.

Twenty-four hours following reperfusion, the rats would bereanesthetized, the heart removed, and the left ventricles cut parallelto the atrioventricular space sulcus into slices. The tissue slices maybe stained at 37° C. for 15 minutes using an appropriate dye,particularly triphenyltetrazolium chloride, and the size of theinfarction determined by weighing the tissue.

It would be expected that the size of infarcts in tissue taken from ratsthat receive the intracellular IL-1 receptor antagonist would bedecreased by about 50% compared to tissue removed from control animals.

Example 5 IL-1 Receptor Antagonist as a Cancer Therapeutic

IL-1 is an autocrine growth factor for certain cancers. Tsai andGaffney, 1987, JNCI, 79:77-81. Thus, the IL-1 receptor antagonistinhibitor would interfere with the autocrine activity of IL-1 and be aneffective chemotherapeutic for the treatment of this form of cancer. Theinhibitor can be administered in an effective amount, with the dosage ofthe inhibitor normally being determined by the prescribing physician. Itis to be expected that the dosage will vary according to the tumor type,tumor mass, as well as the age, weight, and response of the individualpatient. Typically, the amount of inhibitor administered per dose willbe in the range of about 0.1 to 25 mg/kg of body weight, with thepreferred dose being about 0.1 to 10 mg/kg of patient body weight. orparenteral administration, the inhibitor will be formulated in aninjectable form combined with a pharmaceutically acceptable parenteralvehicle. Such vehicles are well known in the art and examples includewater, saline, Ringer's solution, dextrose solution, and solutionsconsisting of small amounts of the human serum albumin. The vehicle maycontain minor amounts of additives that maintain the isotonicity andstability of the inhibitor. The preparation of such solutions is withinthe skill of the art. Typically, the IL-1 receptor antagonist inhibitorwill be formulated in such vehicles at a concentration of about 1-8.0mg/ml.

Example 6 IL-1 Receptor Antagonist as an AIDS Medicament

Using the materials and methods described above or that are well knownin the art, the IL-1 receptor antagonist cDNA (SEQ ID NO:1) presented inFIG. 1 (SEQ ID NO:1) was transfected into a human T-cell line, CEM, andshown to down regulate the expression of CD4 as revealed by FACS usinganti-CD4 antibody. CEM cells transfected with the antagonist had nodetectible CD4 on the cell surface, and such cells when exposed to HIVare not infected by virus as revealed by reverse transcriptase activity(FIG. 2). FIG. 2 also shows that the secreted form of the IL-1 receptorantagonist is not effective in blocking HIV infection, and FACS sortingrevealed that this molecule does not down regulate CD4 expression.

It is apparent, based on the above results, that the intracellular formof the IL-1 receptor antagonist, or the secreted form without its leadersequence, would be useful to prevent or control HIV infection byremoving CD4 positive T-cells from a patient, transfecting the cellswith either antagonist, but preferably the intracellular form, andreturning the cells to the patient. Such cells would no longer besubject to infection by the virus, and thus significantly extend thelife span of the patient.

Example 7 Intracellular IL-1 Receptor Antagonist for the Detection ofEndometrial Cancer

To determine whether alterationS of IL-1 mediated events by receptorantagonists may further be implicated in the pathogenesis of cancer westudied the expression of IL-1 and IL-1 receptor antagonists in normalendometrium and endometrial cancers. RNA was extracted from 11 benignendometrium and 20 endometrial cancers. mRNA expression of IL-1,secreted (sIL-1ra), and intracellular (icIL-1ra) IL-1 receptorantagonist was determined using reverse transcriptase polymerase chainreaction. IL-1 was expressed by normal endometrium and endometrialcancer, expression did not differ between these two tissue types.sIL-1ra was not expressed by any endometrial sample. However, normalsamples showed minimal expression of icIL-1ra while endometrial cancersshowed significantly higher levels of expression, up to 100×(p=0.03).Because icIL-1ra transcripts were increased in endometrial cancers,tissue samples were stained with a polyclonal antibody recognizing IL-1receptor antagonists. Normal endometrium showed epithelial staining andno stromal staining for IL-1ra In contrast, in endometrial cancers thestromal component stained strongly for IL-1ra We conclude that icIL-1ramRNA expression is increased in endometrial cancers and corresponds toincreased IL-1ra seen in stromal tissue in neoplasias.

The present invention has been described with reference to specificembodiments. However, this application is intended to cover thosechanges and substitutions which may be made by those skilled in the artwithout departing from the spirit and the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 2    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 602 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    AGCTCCACCCTGGGAGGGACTGTGGCCCAGGTACTGCCCGGGTGCTACTTTATGGGCAGC60    AGCTCAGTTGAGTTAGAGTCTGGAAGACCTCAGAAGACCTCCTGTCCTATGAGGCCCTCC120    CCATGGCTTTAGAGACGATCTGCCGACCCTCTGGGAGAAAATCCAGCAAGATGCAAGCCT180    TCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTG240    GATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGATAGATGTGGTACCCATTGAGC300    CTCATGCTCTGTTCTTGGGAATCCATGGAGGGAAGATGTGCCTGTCCTGTGTCAAGTCTG360    GTGATGAGACCAGACTCCAGCTGGAGGCAGTTAACATCACTGACCTGAGCGAGAACAGAA420    AGCAGGACAAGCGCTTCGCCTTCATCCGCTCAGACAGTGGCCCCACCACCAGTTTTGAGT480    CTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCC540    TCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACTTCCAGGAGGACGAGT600    AG602    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 159 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetAlaLeuGluThrIleCysArgProSerGlyArgLysSerSerLys    151015    MetGlnAlaPheArgIleTrpAspValAsnGlnLysThrPheTyrLeu    202530    ArgAsnAsnGlnLeuValAlaGlyTyrLeuGlnGlyProAsnValAsn    354045    LeuGluGluLysIleAspValValProIleGluProHisAlaLeuPhe    505560    LeuGlyIleHisGlyGlyLysMetCysLeuSerCysValLysSerGly    65707580    AspGluThrArgLeuGlnLeuGluAlaValAsnIleThrAspLeuSer    859095    GluAsnArgLysGlnAspLysArgPheAlaPheIleArgSerAspSer    100105110    GlyProThrThrSerPheGluSerAlaAlaCysProGlyTrpPheLeu    115120125    CysThrAlaMetGluAlaAspGlnProValSerLeuThrAsnMetPro    130135140    AspGluGlyValMetValThrLysPheTyrPheGlnGluAspGlu    145150155    __________________________________________________________________________

We claim:
 1. A method for reducing the reperfusion injury in a patientfollowing ischemia, comprising the step of administering to a patient inneed of treatment an amount of an IL-1 receptor antagonist according toSEQ ID NO:2, effective to reduce reperfusion injury.
 2. The method ofclaim 1 wherein the IL-1 receptor antagonist is administeredparenterally.
 3. The method of claim 2 wherein the amount of IL-1receptor antagonist is about 0.1 to 25 mg/kg of patient body weight. 4.The method of claim 3 wherein the amount of IL-1 receptor antagonist isabout 0.1 to 10 mg/kg of patient body weight.
 5. A method for treating adisease state resulting from the IL-1 induced adhesion of leukocytes toendothelial cells, comprising the step of administering to a patient inneed of treatment an amount of an IL-1 receptor antagonist according toSEQ ID NO: 2, effective to treat or prevent said disease state.
 6. Amethod as recited in claim 5, wherein said leukocytes are monocytes orneutrophils.
 7. The method according to claim 5, wherein said diseasestate is a cancer for which IL-1 is an autocrine growth factor.
 8. Themethod of claim 5 wherein the IL-1 receptor antagonist is administeredparenterally.
 9. The method of claim 8 wherein the amount of IL-1receptor antagonist is about 0.1 to 25 mg/kg of patient body weight. 10.The method of claim 9 wherein the amount of IL-1 receptor antagonist isabout 0.1 to 10 mg/kg of patient body weight.
 11. A method forinhibiting the IL-1 induced expression of a leukocyte adhesion moleculeby endothelial cells said method comprising contacting said endothelialcells with an amount of an IL-1 receptor antagonist of SEQ ID NO: 2effective to inhibit expression of said adhesion molecule.
 12. Themethod of claim 11 wherein said leukocyte is a monocyte or a neutrophil.13. The method of claim 12 wherein said leukocyte is a monocyte.
 14. Amethod for treating arthritis, comprising administering to a patient inneed of treatment a therapeutically effective amount of an intracellularIL-1 receptor antagonist of SEQ ID NO:
 2. 15. The method of claim 14,wherein the IL-1 receptor antagonist is administered parenterally. 16.The method of claim 15, wherein the amount of IL-1 receptor antagonistadministered is about 0.1 to 25 mg/kg of patient body weight.
 17. Themethod of claim 16, wherein the amount of IL-1 receptor antagonistadministered is about 0.1 to 10 mg/kg of patient body weight.
 18. Themethod of claim 14, wherein the IL-1 receptor antagonist is formulatedat a concentration of about 1-8.0 mg./ml.